Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase

Maschio, L.; Back, C. R.; Alnawah, J.; Bowen, J. I.; Johns, S. T.; Mbatha, S. Z.; Han, L.-C.; Lees, N. R.; Zorn, K.; Stach, J. E. M.; Hayes, M. A.; van der Kamp, M. W.; Pudney, C. R.; Burston, S. G.; Willis, C. L.; Race, P. R.

doi:10.1101/2024.04.18.590041

bioRxiv · 10.1101/2024.04.18.590041

Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase

Maschio, L.·Back, C. R.·Alnawah, J.·Bowen, J. I.·Johns, S. T.·Mbatha, S. Z.·Han, L.-C.·Lees, N. R.·Zorn, K.·Stach, J. E. M.·Hayes, M. A.·van der Kamp, M. W.·Pudney, C. R.·Burston, S. G.·Willis, C. L.·Race, P. R.

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Abstract

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

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Maschio, L., Back, C. R., Alnawah, J., Bowen, J. I., Johns, S. T., Mbatha, S. Z., Han, L.-C., Lees, N. R., Zorn, K., Stach, J. E. M., Hayes, M. A., van der Kamp, M. W., Pudney, C. R., Burston, S. G., Willis, C. L., Race, P. R.. 2024-04-18. Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase. https://doi.org/10.1101/2024.04.18.590041

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